COMPOSITIONS AND METHODS FOR siRNA INHIBITION OF ANGIOGENESIS

ABSTRACT

RNA interference using small interfering RNAs which are specific for the vascular endothelial growth factor (VEGF) gene and the VEGF receptor genes Flt-1 and Flk-1/KDR inhibit expression of these genes. Diseases which involve angiogenesis stimulated by overexpression of VEGF, such as diabetic retinopathy, age related macular degeneration and many types of cancer, can be treated by administering the small interfering RNAs.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/294,228filed on Nov. 14, 2002, which claims the benefit of U.S. provisionalpatent application Ser. No. 60/398,417, filed on Jul. 24, 2002.

JOINT RESEARCH AGREEMENT

Not Applicable

REFERENCE TO GOVERNMENT GRANT

The invention described herein was supported in part by NIH/NEI grantno. R01-EY10820, EY-13410 and EY12156. The U.S. government has certainrights in this invention.

FIELD OF THE INVENTION

This invention relates to the regulation of gene expressing by smallinterfering RNA, in particular for treating disease or conditionsinvolving angiogenesis.

BACKGROUND OF THE INVENTION

Angiogenesis, defined as the growth of new capillary blood vessels or“neovascularization,” plays a fundamental role in growth anddevelopment. In mature humans, the ability to initiate angiogenesis ispresent in all tissues, but is held under strict control, a keyregulator of angiogenesis is vascular endothelial growth factor(“VEGF”), also called vascular permeability factor (“VPF”). VEGF existsin at least four different alternative splice forms in humans (VEGF₁₂₁,VEGF₁₆₅, VEGF₁₈₉ and VEGF₂₀₆), all of which exert similar biologicalactivities.

Angiogenesis is initiated when secreted VEGF binds to the Flt-1 andFlk-1/KDR receptors (also called VEGF receptor 1 and VEGF receptor 2),which are expressed on the surface of endothelial cells. Flt-1 andFlk-1/KDR are transmembrane protein tyrosine kinases, and binding ofVEGF initiates a cell signal cascade resulting in the ultimateneovascularization in the surrounding tissue.

Aberrant angiogenesis, or the pathogenic growth of new blood vessels, isimplicated in a number of conditions. Among these conditions arediabetic retinopathy, psoriasis, exudative or “wet” age-related maculardegeneration (“ARMD”), rheumatoid arthritis and other inflammatorydiseases, and most cancers. The diseased tissues or tumors associatedwith these conditions express abnormally high levels of VEGF, and show ahigh degree of vascularization or vascular permeability.

ARMD in particular is a clinically important angiogenic disease. Thiscondition is characterized by choroidal neovascularization in one orboth eyes in aging individuals, and is the major cause of blindness inindustrialized countries.

A number of therapeutic strategies exist for inhibiting aberrantangiogenesis, which attempt to reduce the production or effect of VEGF.For example, anti-VEGF or anti-VEGF receptor antibodies (Kim E S et al.(2002), PNAS USA 99: 11399-11404), and soluble VEGF “traps” whichcompete with endothelial cell receptors for VEGF binding (Holash J etal. (2002), PNAS USA 99: 11393-11398) have been developed. ClassicalVEGF “antisense” or aptamer therapies directed against VEGF geneexpression have also been proposed (U.S. published application2001/0021772 of Uhlmann et al.). However, the anti-angiogenic agentsused in these therapies can produce only a stoichiometric reduction inVEGF or VEGF receptor, and the agents are typically overwhelmed by theabnormally high production of VEGF by the diseased tissue. The resultsachieved with available anti-angiogenic therapies have therefore beenunsatisfactory.

RNA interference (hereinafter “RNAi”) is a method ofpost-transcriptional gene regulation that is conserved throughout manyeukaryotic organisms. RNAi is induced by short (i.e. <30 nucleotide)double stranded RNA (“dsRNA”) molecules which are present in the cell(Fire A et al. (1998). Nature 391: 806-811). These short dsRNA moleculescalled “short interfering RNA” or “siRNA,” cause the destruction ofmessenger RNAs (“mRNAs”) which share sequence homolog) with the siRNA towithin one nucleotide resolution (Elbashir S M et al. (2001), Genes Dev,15 188-200). It is believed that the siRNA and the targeted mRNA bind toan “RNA-induced silencing complex” or “RISC”, which cleaves the targetedmRNA. The siRNA is apparently recycled much like a multiple-turnoverenzyme, with 1 siRNA molecule capable of inducing cleavage ofapproximately 1000 mRNA molecules. siRNA-mediated RNAi degradation of anmRNA is therefore more effective than currently available technologiesfor inhibiting expression of a target gene.

Elbashir S M et al. (2001), supra, has shown that synthetic siRNA of 21and 22 nucleotides in length, and which have short 3′ overhangs, areable to induce RNAi of target mRNA in a Drosophila cell lysate. Culturedmammalian cells also exhibit RNAi degradation with synthetic siRNA(Elbashir S M et al. (2001) Nature 411: 494-498), and RNAi degradationinduced by synthetic siRNA has recently been shown in living mice(MeCaffrey A P et al . (2002), Nature, 418: 38-49; Xia H et al. (2002),Nat. Biotech. 20: 1006-1010). The therapeutic potential of siRNA-inducedRNAi degradation has been demonstrated in several recent in vitrostudies, including the siRNA-directed inhibition of HIV-1 infection(Novina CD et al. (2002), Nat. Med. 8: 681-686) and reduction ofneurotoxic polyglutamine disease protein expression (Xia H et al.(2002), supra).

What is needed, therefore, are agents which selectively inhibitexpression of VEGF or VEGF receptors in catalytic or sub-stoichiometricamounts.

SUMMARY OF THE INVENTION

The present invention is directed to siRNAs which specifically targetand cause RNAi-induced degradation of mRNA from VEGF, Flt-1 andFlk-1/KDR genes. The siRNA compounds and compositions of the inventionare used to inhibit angiogenesis, in particular for the treatment ofcancerous tumors, age-related macular degeneration, and other angiogenicdiseases.

Thus, the invention provides an isolated siRNA which targets human VEGFmRNA, human Flt-1 mRNA, human Flk-1/KDR mRNA, or an alternative spliceform, mutant or cognate thereof. The siRNA comprises a sense RNA strandand antisense RNA strand which form an RNA duplex. The sense RNA strandcomprises a nucleotide sequence identical to a target sequence of about19 to about 25 contiguous nucleotides in the target mRNA.

The invention also provides recombinant plasmids and viral vectors whichexpress the siRNA of the invention, as well as pharmaceuticalcompositions comprising the siRNA of the invention and apharmaceutically acceptable carrier.

The invention further provides a method of inhibiting expression ofhuman VEGF mRNA, human Flt-1 mRNA, human Flk-1/KDR mRNA, or canalternative splice form, mutant or cognate thereof, comprisingadministering to a subject an effective amount of the siRNA of theinvention such that the target mRNA is degraded.

The invention further provides a method of inhibiting angiogenesis in asubject, comprising administering to a subject an effective amount of ansiRNA targeted to human VEGF mRNA, human Flt-1 mRNA, human Flk-1/KDRmRNA, or an alternative splice form, mutant or cognate thereof.

The invention further provides a method of treating an angiogenicdisease comprising administering to a subject in need of such treatmentan effective amount of an siRNA targeted to human VEGF mRNA, human Flt-1mRNA, human Flk-1/KDR mRNA, or an alternative splice form, mutant orcognate thereof, such that angiogenesis associated with the angiogenicdisease is inhibited.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are a histograms of VEGF concentration (in siRNA(nonspecific); or siRNA targeting human VEGF mRNA (“VEGF”). VEGFconcentration (in pg/ml) in non-hypoxic 293 and HeLa cells is alsoshown. Each bar represents the average of four experiments, and theerror is the standard deviation of the mean.

FIG. 2 is a histogram of murine VEGF concentration (in pg/ml) in hypoxicNIH 3T3 cells treated with no siRNA (“−”); nonspecific siRNA(“nonspecific”); or siRNA targeting human VEGF mRNA (“VEGF”). Each barrepresents the average of six experiments and the error is the standarddeviation of the mean.

FIG. 3 is a histogram of human VEGF concentration (pg/total protein) inretinas from mice injected with adenovirus expressing human VEGF(“AdVEGF”) in the presence of either GFP siRNA (dark gray bar) or humanVEGF siRNA (light gray bar). Each bar represents the average of 5 eyesand the error bars represent the standard error of the mean.

FIG. 4 is a histogram showing the mean are (in mm²) of laser-induced CNVin control eyes given subretinal injections of GFP siRNA (N=9; “GFPsiRNA”), and in eyes given subretinal injections of mouse VEGF siRNA(N=7; “Mouse VEGF siRNA”). The error bars represent the standard errorof the mean.

FIG. 5 is a schematic representation of pAAVsiRNA, a cis-acting plasmidused to generate a recombinant AAV viral vector of the invention. “ITR”;AAV inverted terminal repeats; “U6”; U6 RNA promoters; “Sense”: siRNAsense coding sequence, “Anti”; siRNA antisense coding sequence; “PolyT”:polythymididne termination signals.

FIG. 6 shows histograms of the mean area (in mm²) of laser-induced CNVin treatment in mouse eyes injected (A) subretinally or (B)intravitreally with a mouse anti-VEGF siRNA (“mVEGF1.siRNA”) or controlsiRNA (“GFP1.siRNA”). The error bars represent the standard error of themean. (C) is a histogram of the mean area (in mm²) of laser-induced CNVin mouse eyes injected intravitreally with: phosphate-buffered salinewith no siRNA at 1 day post-laser induction (“PBS”; CNV area measured at14 days post-laser induction); control siRNA at 14 days post-laserinduction (“GFP1.siRNA”; CNV area measured at 21 days post-laserinduction); or a mouse anti-VEGF siRNA at 14 days post-laser induction(“mVEGF1.siRNA”; CNV area measured at 21 days post-laser induction). Theerror bars represent the standard error of the mean.

FIG. 7 is a graph of the percent of VEGF (“%VEGF”) protein in mouse eyesinjected sub-retinally with human anti-VEGF siRNA (“Cand5”) and controlsiRNA (“GFP1.siRNA”) at 0 (n=2; pre-siRNA injection) 6 (n=3), 10 (n=3)and 14 (n=3) days post-injection. %VEGF=([VEGF] in the Cand5 eye/[VEGF]in the GFP1.siRNA eye) * 100.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise indicated, all nucleic acid sequences herein are givenin the 5′ to 3′ direction. Also, all deoxyribonucleotides in a nucleicacid sequence are represented by capital letters (e.g. deoxythymidine is“T”), and ribonucleotides in a nucleic acid sequence are represented bylower case letters (e.g. uridine is “u”).

Compositions and methods comprising, siRNA targeted to VEGF, Flt-1 orFlk-1/KDR mRNA are advantageously used to inhibit angiogenesis, inparticular for the treatment of angiogenic disease. The siRNA of theinvention are believed to cause the RNAi-mediated degradation of thesemRNAs, so that the protein product of the VEGF, Flt-1 or Flk-1/KDR genesis not produced or is produced in reduced amounts. Because VEGF bindingto the Flt-1 or Flk-1/KDR receptors is required for initiating andmaintaining angiogenesis, the siRNA-mediated degradation of VEGF, Flt-1or Flk-1/KDR mRNA inhibits the angiogenic process.

The invention therefore provides isolated siRNA comprising shortdouble-stranded RNA from about 17 nucleotides to about 29 nucleotides inlength, preferably from about 19 to about 25 nucleotides in length, thatare targeted to the target mRNA. The siRNA comprise a sense RNA strandand a complementary antisense RNA strand annealed together by standardWatson-Crick base-pairing interactions (hereinafter “base-paired”). Asis described in more detail below, the sense strand comprises a nucleicacid sequence which is identical to a target sequence contained withinthe target mRNA.

The sense and antisense strands of the present siRNA can comprise twocomplementary, single-stranded RNA molecules or can comprise a singlemolecule in which two complementary portions are base-paired and arecovalently linked by a single-stranded “hairpin” area. Without wishingto be bound by any theory, it is believed that the hairpin area of thelatter type of siRNA molecule is cleaved intracellularly by the “Dicer”protein (or its equivalent) to form an siRNA of two individualbase-paired RNA molecules (see Tuschl, T. (2002), supra).

As used herein, “isolated” means altered or removed from the naturalstate through human intervention. For example, an siRNA naturallypresent in a living animal is not “isolated,” but a synthetic siRNA, oran siRNA partially or completely separated from the coexisting materialsof its natural state is “isolated.” An isolated siRNA can exist insubstantially purified form, or can exist in a non-native environmentsuch as, for example, a cell into which the siRNA has been delivered.

As used herein, “target mRNA” means human VEGF, Flt-1 or Flk-1/KDR mRNA,mutant or alternative splice forms of human VEGF, Flt-1 or Flk-1/KDRmRNA or mRNA from cognate VEGF, Flt-1 or Flk-1/KDR genes.

As used herein, a gene or mRNA which is “cognate” to human VEGF, Flt-1or Flk-1/KDR is a gene or mRNA from another mammalian species which ishomologous to human VEGF, Flt-1 or Flk-1/KDR. For example, the cognateVEGF mRNA from the mouse is given in SEQ ID NO: 1.

Splice variants of human VEGF are known, including VEGF₁₂₁(SEQ ID NO:2), VEGF₁₆₅ (SEQ ID NO: 3), VEGF₁₈₉ (SEQ ID NO: 4) and VEGF₂₀₆ (SEQ IDNO: 5). The mRNA transcribed from the human VEGF, Flt-1 (SEQ ID NO: 6)or Flk-1/KDR (SEQ ID NO: 7 ) genes can be analyzed for furtheralternative splice, forms using techniques well-known in the art. Suchtechniques include reverse transcription-polymerase chain reaction(RT-PCR), northern blotting and in-situ hybridization. Techniques foranalyzing mRNA sequences are described, for example, in Busting S A(2000), J. Mol. Endocrinol. 25: 169-193, the entire, disclosure of whichis herein incorporated by reference. Representative techniques foridentifying alternatively spliced mRNAs are also described below.

For example databases that contain nucleotide sequences related to agiven disease gene can be used to identify alternatively spliced mRNA.Such databases include GenBank, Embase, and the Cancer Genome AnatomyProject (CGAP) database. The CGAP database, for example, containsexpressed sequence tags (ESTs) from various types of human cancers. AnmRNA or gene sequence from the VEGF, Flt-1 or Flk-1/KDR genes can beused to query such a database to determine whether ESTs representingalternatively spiced mRNAs have been found for a these genes.

A technique called “RNAse protection” can also be used to identifyalternative spliced VEGF, Flt-1 or Flk-1/KDR mRNAs. RNAse protectioninvolves translation of a gene sequence into synthetic RNA, which ishybridized to RNA derived from other cells; for example, cells fromtissue at or near the site of neovascularization. The hybridized RNA isthen incubated with enzymes that recognize RNA:RNA hybrid mismatches.Smaller than expected fragments indicate the presence of alternativelyspliced mRNAs. The putative alternatively spliced mRNAs can be clonedand sequenced by methods well known to those skilled in the art.

RT-PCR can also be used to identify alternatively spliced VEGF, Flt-1 orFlk-1/KDR mRNAs. In RT-PCR, mRNA from the diseased tissue is convertedinto cDNA by the enzyme reverse transcriptase, using methods well-knownto those of ordinary skill in the art. The entire coding sequence of thecDNA is then amplified via PCR using a forward primer located in the 3′untransulated region, and a reverse primer located in the 5′untranslated region. The amplified products can be analyzed foralternative splice forms, for example by comparing the size of theamplified products with the size of the expected product from normallyspliced mRNA, e.g., agarose gel electrophotesis. Any change in the sizeof the amplified product can indicate alternative splicing.

mRNA produced from mutant VEGF, Flt-1 or Flk-1/KDR genes can also bereadily identified through the techniques described above foridentifying alternative splice forms. As used herein, “mutant ” VEGF,Flt-1 or Flk-1/KDR genes or mRNA include human VEGF, Flt-1 or Flk-1/KDRgenes or mRNA which differ in sequence from the VEGF Flt-1 or Flk-1/KDRsequences set forth herein. Thus, alleic forms of these genes, and themRNA produced from them, are considered “mutants” for purposes of thisinvention.

It is understood that human VEGF, Flt-1 or Flk-1/KDR mRNA may containtarget sequences in common with their respective alternative spliceforms, cognates or mutants. A single mRNA comprising such a commontargeting sequence can therefore induce RNAi-mediated degradation ofdifferent RNA types which contain the common targeting sequence.

The siRNA of the invention can comprise partially purified RNAsubstantially pure RNA, synthetic RNA, or recombinantly produced RNA, aswell as altered RNA that differs from naturally-occurring RNA by theaddition, deletion, substitution and/or alteration of one or morenucleotides. Such alterations can include addition of non-nucleotidematerial, such as to the end(s) of the siRNA or to one or more internalnucleotides of the siRNA, including modifications that make the siRNAresistant to nuclease digestion.

One or both strands of the siRNA of the invention can also comprise a 3′overhang. As used herein, a “3′ overhang” refers to at least oneunpaired nucleotide extending from the 3′-end of a duplexed RNA strand.

Thus in one embodiment, the siRNA of the invention comprises at leastone 3′ overhang of from 1 to about 6 nucleotides (which includesribonucleotides or deoxynucleotides) in length, preferably from 1 toabout 5 nucleotides in length, more preferably from 1 to about 4nucleotides in length, and particularly preferably from about 2 to about4 nucleotides in length.

In the embodiment in which both strands of the siRNA molecule comprise a3′ overhang, the length of the overhangs can be the same or differentfor each strand. In a most preferred embodiment, the 3′ overhang ispresent on both strands of the siRNA, and is 2 nucleotides in length.For example, each strand of the siRNA of the invention can comprise 3′overhangs of dithymidylic acid (“TT”) or diuridylci acid (“uu”).

In order to enhance the stability of the present siRNA, the 3′ overhangscan be also stabilized against degradation. In one embodiment, theoverhangs are stabilized by including purine nucleotides, such asadenosine or guanosine nucleotides. Alternatively, substitution ofpyrimidine nucleotides by modified analogues, e.g., substitution ofuridine nucleotides in the 3′ overhangs with 2′-deoxythymidine, istolerated and does not affect the efficiency of RNAi degradation. Inparticular, the absence of a 2′ hydroxyl in the 2′-deoxythymidinesignificantly enhances the nuclease resistance of the 3′ overhang intissue culture medium.

In certain embodiments, the siRNA of the invention comprises thesequence AA(N19)TT or NA(N21), where N is any nucleotide. These siRNAcomprise approximately 30-70% GC, and preferably comprise approximately50% G/C. The sequence of the siRNA strand corresponds to (N19)TT or N21(i.e., position is 3 to 23), respectively. In the latter case, the 3′end of the sense siRNA is converted to TT. The rationale for thissequence conversion is to generate a symmetric duplex with respect tothe sequence composition of the sense and antisense strand 3′ overhangs.The antisense RNA strand is then synthesized as the complement topositions 1 to 21 of the sense strand.

Because position 1 of the 23-nt sense strand in these embodiments is notrecognized in a sequence-specific manner by the antisense strand, the3′-most nucleotide residue of the antisense strand can be, chosendeliberately. However, the penultimate nucleotide of the antisensestrand (complementary to position 2 of the 23-nt sense strand in eitherembodiment) is generally complementary to the targeted sequence.

In another embodiment, the siRNA of the invention comprises the sequenceNAR(N17)YNN, where R is a purine (e.g., A or G) and Y is a pyrimidine(e.g., C or U/T). The respective 21-nt sense and antisense RNA strandsof this embodiment therefore generally begin with a purine nucleotide.Such siRNA can be expressed from pol III expression vectors without achange in targeting site, as expression of RNAs from pol III promotersis only believed to be efficient when the first transcribed nucleotideis a purine.

The siRNA of the invention can be targeted to any stretch ofapproximately 19-25 contiguous nucleotides in any of the target mRNAsequences (the “target sequence”). Techniques for selecting targetsequences for siRNA are given, for example, in Tuschl T et al., “ThesiRNA User Guide,” revised Oct. 11, 2002, the entire disclosure of whichis herein incorporated by reference. “The siRNA User Guide” is availableon the world wide web at a website maintained by Dr. Thomas Tuschl,Department of Cellular Biochemistry, AG 105, Max-Planck-Institute forBiophysical Chemistry, 37077 Göttingen, Germany, and can be found byaccessing the website of the Max Planck Institute and searching with thekeyword “siRNA.” Thus, the sense strand of the present siRNA comprises anucleotide sequence identical to any contiguous stretch of about 19 toabout 25 nucleotides in the target mRNA.

Generally, a target sequence on the target mRNA can be selected from agiven cDNA sequence corresponding to the target mRNA, preferablybeginning 50 to 100 nt downstream (i.e., in the 3′ direction) from thestart codon. The target sequence can, however, be located in the 5′ or3′ untranslated regions, or in the region nearby the start codon (seee.g., the target sequences of SEQ ID NOS: 73 and 74 in Table 1 below,which are within 100 nt of the 5′-end of the VEGF₁₂₁ cDNA

For example, a suitable target sequence in the VEGF₁₂₁ cDNA sequence is:TCATCACGAAGTGGTGAAG (SEQ ID NO: 8)

Thus, an siRNA of the invention targeting this sequence, and which has3′ uu overhangs on each strand (overhangs shown in bold), is:5′-ucaucacgaaguggugaaguu-3′ (SEQ ID NO: 9)3′-uuaguagugcuucaccacuuc-5′ (SEQ ID NO: 10)

An siRNA of the invention targeting this same sequence, but having 3′ TToverhangs on each strand (overhangs shown in bold) is;5′-ucaucacgaaguggugaagTT-3′ (SEQ ID NO: 11)3′-TTaguagugcuucaccacuuc-5′ (SEQ ID NO: 12)

Other VEGF₁₂₁ target sequences from which siRNA of the invention can bederived are given in Table 1. It is understood that all VEGF₁₂₁ targetsequences listed in Table 1 are within that portion of the VEGF₁₂₁alternative splice form which is common to all human VEGF alternativesplice forms. Thus, the VEGF₁₂₁ target sequences in Table 1 can alsotarget VEGF₁₆₅, VEGF₁₈₉ and VEGF₂₀₆ mRNA. Target sequences which targeta specific VEGF isoform can also be readily identified. For example, atarget sequence which targets VEGF₁₆₅ mRNA but not VEGF₁₂₁ mRNA isAACGTACTTGCAGATGTGACA (SEQ ID NO: 13). Exemplary target sequences forhuman Flt-1 are given in SEQ ID NOS: 91-504, and exemplary targetsequences for human Elk-1/KDR are given in SEQ ID NOS: 505-864. TABLE 1VEGF Target Sequences SEQ SEQ target sequence ID NO: target sequence IDNO: GTTCATGGATGTCTATCAG 14 TCCCTGTGGGCCTTCCTCA 30 TCGAGACCCTGGTGGACAT 15GCATTTGTTTGTACAAGAT 31 TGACGAGGGCCTGGAGTGT 16 GATCCGCAGACGTGTAAAT 32TGACGAGGGCCTGGAGTGT 17 ATGTTCCTGCAAAAACACA 33 CATCACCATGCAGATTATG 18TGTTCCTGCAAAAACACAG 34 ACCTCACCAAGGCCAGCAC 19 AAACACAGACTCGCGTTGC 35GGCCAGCACATAGGAGAGA 20 AACACAGACTCGCGTTGCA 36 CAAATGTGAATGCAGACCA 21ACACAGACTCGCGTTGCAA 37 ATGTGAATGCAGACCAAAG 22 CACAGACTCGCGTTGCAAG 38TGCAGACCAAAGAAAGATA 23 GGCGAGGCAGCTTGAGTTA 39 AGAAAGATAGAGCAAGACA 24ACGAACGTACTTGCAGATG 40 GAAAGATAGAGCAAGACAA 25 CGAACGTACTTGCAGATGT 41GATAGAGCAAGACAAGAAA 26 CGTACTTGCAGATGTGACA 42 GACAAGAAAATCCCTGTGG 27GTGGTCCCAGGCTGCACCC 43 GAAAATCCCTGTGGGCCTT 28 GGAGGAGGGCAGAATCATC 44AATCCCTGTGGGCCTTGCT 29 GTGGTGAAGTTCATGGATG 45 AATCATCACGAAGTGGTGAAG 46AAGCATTTGTTTGTACAAGATCC 62 AAGTTCATGGATGTCTATCAG 47AAGATCCGCAGACGTGTAAATGT 63 AATCGAGACCCTGGTGGACAT 48AAATGTTCCTGCAAAAACACAGA 64 AATGACGAGGGCGTGGAGTGT 49AATGTTCCTGCAAAAACACAGAC 65 AACATCACCATGCAGATTATG 50AAAAACACAGACTCGCGTTGCAA 66 AAACCTCACCAAGGCCAGCAC 51AAAACACAGACTCGCGTTGCAAG 67 AAGGCCAGCACATAGGAGAGA 52AAACACAGACTCGCGTTGCAAGG 68 AACAAATGTGAATGCAGACCA 53AACACAGACTCGCGTTGCAAGGC 69 AAATGTGAATGCAGACCAAAG 54AAGGCGAGGCAGCTTGAGTTAAA 70 AATGCAGACCAAAGAAAGATA 55AAACGAACGTACTTGCAGATGTG 71 AAAGAAAGATAGAGCAAGACA 56AACGAACGTACTTGCAGATGTGA 72 AAGAAAGATAGAGCAAGACAA 57AAGTGGTCCCAGGCTGCACCCAT 73 AAGATAGAGCAAGACAAGAAAAT 58AAGGAGGAGGGCAGAATCATCAC 74 AAGACAAGAAAATCCCTGTGGGC 59AAGTGGTGAAGTTCATGGATGTC 75 AAGAAAATCCCTGTGGGCCTTGC 60AAAATCCCTGTGGGCCTTGCTCA 76 AATCCCTGTGGGCCTTGCTCAGA 61GGCAGAATCATCACGAAGTGG 81 CCTGGTGGACATCTTCCAGGA 82 CACACACTCGCGTTGCAAGGC87 GAGATCGAGTACATCTTCAAG 83 TCACCATGCAGATTATGCGGA 88TGGAGTGTGTGCCCACTGAGG 84 TAGAGCAAGACAAGAAAATCC 89 GAGCTTCCTACAGCACAACAA85 CCGCAGACGTGTAAATGTTCC 90 TTGCTCAGAGCGGAGAAAGCA 86

The siRNA, of the invention can be obtained using a number of techniquesknown to those of skill in the art. For example, the siRNA can bechemically synthesized or recombinantly produced using methods known inthe art, such as the Drosophila in vitro: system described in U.S.published application 2002/0086356 of Tuschl et al. the entiredisclosure of which is herein incorporated by reference.

Preferably, the siRNA of the invention are chemically synthesized usingappropriately protected ribonucleotides phosphoramidites and aconventional DNA/RNA synthesizer. The siRNA can be synthesized as twoseparate, complementary RNA molecules, or as a single RNA molecule withtwo complementary regions. Commercial suppliers of synthetic RNAmolecules or synthesis reagents include Proligo (Hamburg, Germany),Dharmacon Research (Lafayette, Colo. USA), Pierce Chemical (part ofPerbio Science, Rockford, Ill., USA), Glen Research (Sterling, Va. USA),ChemGenes (Ashland, Mass. USA) and Cruachem (Glasgow, UK).

Alternatively, siRNA can also be expressed from recombinant circular orlinear DNA plasmids using any suitable promoter. Suitable promoters forexpressing siRNA of the invention from a plasmid include, for example,the U6 or H1 RNA pol III promoter sequences and the cytomegaloviruspromoter. Selection of other suitable promoters is within the skill inthe art. The recombinant plasmids of the invention can also compriseinducible or regulatable promoters for expression of the siRNA in aparticular tissue or in a particular intracellular environment.

The siRNA expressed from recombinant plasmids can either be isolatedfrom cultured cell expression systems by standard techniques, or can beexpressed intracellularly at or near the area of neovascularization invitro. The use of recombinant plasmids to deliver siRNA of the inventionto cells in vitro is discussed in more detail below.

siRNA of the invention can be expressed from a recombinant plasmideither as two separate, complementary RNA molecules, or as a single RNAmolecule with two complementary regions.

Selection of plasmids suitable for expressing siRNA of the invention,methods for inserting nucleic acid sequences for expressing the siRNAinto the plasmid, and methods of delivering the recombinant plasmid tothe cells of interest are within the skill in the art. See, for example,Tuschl, T. (2002), Nat. Biotechnol, 20: 446-448, Brummelkamp T R et al.(2002), Science 296: 550-553 Miyagishi M et al. (2002), Nat. Biotechnol.20: 497-500; Paddison P J et al. (2002). Genes Dev. 16: 948-9578; Lee NS et al. (2002) Nat. Biotechnol, 20: 500-505; and Paul C P et al.(2002). Nat. Biotechnol. 20: 505-508, the entire disclosures of whichare herein incorporated by reference.

A plasmid comprising nucleic acid sequences for expressing an siRNA ofthe invention is described in Example 7 below. That plasmid, calledpAAVsiRNA, comprises a sense RNA strand coding sequence in operableconnection with a polyT termination sequence under the control of ahuman U6 RNA promoter, and an antisense RNA strand coding sequence inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter. The plasmid pAAVsiRNA is ultimately intendedfor use in producing an recombinant adeno-associated viral vectorcomprising the same nucleic acid sequences for expressing an siRNA ofthe invention.

As used herein, “in operable connection with a polyT terminationsequence” means that the nucleic acid sequences encoding the sense orantisense strands are immediately adjacent to the polyT terminationsignal in the 5′ direction. During transcription of the sense orantisense sequences from the plasmid, the polyT termination signals actto terminate transcription.

As used herein, “under the control” of a promoter means that the nucleicacid sequences encoding the sense or antisense strands are located 3′ ofthe promoter, so that the promoter can initiate transcription of thesense or antisense coding sequences.

The siRNA of the invention can also be expressed from recombinant viralvectors intracellularly at or near the area of neovascularization invivo. The recombinant viral vectors of the invention comprise sequencesencoding the siRNA of the invention and any suitable promoter forexpressing the siRNA sequences. Suitable promoters include, for example,the U6 or H1 RNA pol III promoter sequences and the cytomegaloviruspromoter. Selection of other suitable promoters is within the skill inthe art. The recombinant viral vectors of the invention can alsocomprise inducible or regulatable promoters for expression of the siRNAin a particular tissue or in a particular intracellular environment. Theuse of recombinant viral vectors to deliver siRNA of the invention tocells in vitro is discussed in more detail below.

siRNA of the invention can be expressed from a recombinant viral vectoreither as two separate, complementary RNA molecules, or as a single RNAmolecule with two complementary regions.

Any viral vector capable of accepting the coding sequences for the siRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g.lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of the viral vectors can also be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses. For example, an AAV vector of the inventioncan be pseudotyped with surface proteins from vesicular stomatitis virus(VSV), rabies, Ebola, Mokola, and the like.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe siRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;and Anderson W F (1998), Nature 392: 25-30, the entire disclosures ofwhich are herein incorporated by reference.

Preferred viral vectors are those derived from AV and AAV. In aparticularly preferred embodiment, the siRNA of the invention isexpressed as two separate, complementary single-stranded RNA moleculesfrom a recombinant AAV vector comprising, for example, either the U6 orH1 RNA promoters, or the cytomegalovirus (CMV) promoter.

A suitable AV vector for expressing the siRNA of the invention, a methodfor constructing the recombinant AV vector, and a method for deliveringthe vector into target cells are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the siRNA of the invention, methodsfor constructing the recombinant AAV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol., 70:520-532; Samulski R et al. (1989). J. Virol. 63: 3822-3826; U.S. Pat.No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent ApplicationNo. WO 94/13788; and International Patent Application No. WO 93/24641,the entire disclosures of which are herein incorporated by reference. Anexemplary method for generating a recombinant AAV vector of theinvention is described in Example 7 below.

The ability of an siRNA containing a given target sequence to causeRNA-mediated degradation of the target mRNA can be evaluated usingstandard techniques for measuring the levels of RNA or protein in cells.For example, siRNA of the invention can be delivered to cultured cells,and the levels of target mRNA can be measured by Northern blot or dotblotting techniques, or by quantitative RT-PCR. Alteratively, the levelsof VEGF, Flt-1 or Flk-1/KDR receptor protein in the cultured cells canbe measured by ELISA or Western blot. A suitable cell culture system formeasuring the effect of the present siRNA on target mRNA or proteinlevels is described in Example 1 below.

RNAi-mediated degradation of target mRNA by an siRNA containing a giventarget sequence can also be evaluated with animal models ofneovascularization, such as the ROP or CNV mouse models, For example,areas of neovascularization in an ROP or CNV mouse can be measuredbefore and after administration of an siRNA. A reduction in the areas ofneovascularization in these models upon administration of the siRNAindicates the down-regulation of the target mRNA (see Example 6 below).

As discussed above, the siRNA of the invention target and cause theRNAi-mediated degradation of VEGF, Flt-1 or Flk-1/KDR mRNA, oralternative splice forms, mutants or cognates thereof. Degradation ofthe target mRNA by the present siRNA reduces the production of afunctional gene product from the VEGF, Flt-1 or Flk-1/KDR genes. Thus,the invention provides a method of inhibiting expression of VEGF, Flt-1or Flk-1/KDR in a subject, comprising administering an effective amountof an siRNA of the invention to the subject, such that the target mRNAis degraded. As the products of the VEGF, Flt-1 and Flk-1/KDR genes arerequired for initiating and maintaining angiogenesis, the invention alsoprovides a method of inhibiting angiogenesis in a subject by theRNAi-mediated degradation of the target mRNA by the present siRNA.

As used herein, a “subject” includes a human being or non-human animal.Preferably, the subject is a human being.

As used herein, an “effective amount” of the siRNA is an amountsufficient to cause RNAi-mediated degradation of the target mRNA, or anamount sufficient to inhibit the progression of angiogenesis in asubject.

RNAi-mediated degradation of the target mRNA can be detected bymeasuring levels of the target mRNA or protein in the cells of asubject, using standard techniques for isolating and quantifying mRNA orprotein as described above.

Inhibition of angiogenesis can be evaluated by directly measuring theprogress of pathogenic or nonpathogenic angiogenesis in a subject; forexample, by observing the size of a neovascularized area before andafter treatment with the siRNA of the invention. An inhibition ofangiogenesis is indicated if the size of the neovascularized area staysthe same or is reduced. Techniques for observing and measuring the sizeof neovascularized areas in a subject are within the skill in the art;for example, areas of choroid neovascularization can be observed byophthalmoscopy.

Inhibition of angiogenesis can also be inferred through observing achange or reversal in a pathogenic condition associated with theangiogenesis. For example, in ARMD, a slowing, halting or reversal ofvision loss indicates an inhibition of angiogenesis in the choroid. Fortumors, a slowing, halting or reversal of tumor growth, or a slowing orhalting of tumor metastasis, indicates an inhibition of angiogenesis ator near the tumor site. Inhibition of non-pathogenic angiogenesis canalso be inferred from, for example, fat loss or a reduction incholesterol levels upon administration of the siRNA of the invention.

It is understood that the siRNA of the invention can degrade the targetmRNA (and thus inhibit angiogenesis) in substoichiometric amounts.Without wishing to be bound by any theory, it is believed that the siRNAof the invention causes degradation of the target mRNA in to catalyticmanner. Thus, compared to standard anti-angiogenic therapies,significantly less siRNA needs to be delivered at or near the site ofneovascularization to have a therapeutic effect.

One skilled in the art can readily determine an effective amount of thesiRNA of the invention to be administered to a given subject, by takinginto account factors such as the size and weight of the subject; theextent of the neovascularization or disease penetration; the age, healthand sex of the subject; the route of administration; and whether theadministration is regional or systemic. Generally, an effective amountof the siRNA of the invention comprises an intercellular concentrationat or near the neovascularization site of from about 1 nanomolar (nM) toabout 100 nM, preferably from about 2 nM to about 50 nM, more preferablyfrom about 2.5 nM to about 10 nM. It is contemplated that greater orlesser amounts of siRNA can be administered.

The present methods can be used to inhibit angiogenesis which isnon-pathogenic; i.e., angiogenesis which results from normal processesin the subject. Examples of non-pathogenic angiogenesis includeendomietrial neovascularization, and processes involved in theproduction of fatty tissues or cholesterol. Thus, the invention providesa method for inhibiting non-pathogenic angiogenesis e.g., forcontrolling weight or promoting fat loss, for reducing cholesterollevels, or as an abortifacient.

The present methods can also inhibit angiogenesis which is associatedwith an angiogenic disease; i.e., a disease in which pathogenicity isassociated with inappropriate or uncontrolled antiogenesis. For example,most cancerous solid tumors generate an adequate blood supply forthemselves by inducing angiogenesis in and around the tumor site. Thistumor-induced angiogenesis is often required for tumor growth and alsoallows metastatic cells to enter the bloodstream.

Other angiogenic diseases include diabetic retinopathy, age-relatedmacular degeneration (ARMD), psoriasis, rheumatoid arthritis and otherinflammatory diseases. These diseases are characterized by thedestruction of normal tissue by newly formed blood vessels in the areaof neovascularization. For example, in ARMD, the choroid is invaded anddestroyed by capillaries. The angiogenesis-driven destruction of thechoroid in ARMD eventually leads to partial or full blindness.

Preferably, an siRNA of the invention is used to inhibit the growth ormetastasis of solid tumors associated with cancers; for example breastcancer, lung cancer, head and neck cancer, brain cancer, abdominalcancer, colon cancer, colorectal cancer, esophagus cancer,gastrointestinal cancer, glioma, liver cancer, tongue cancer,neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, retinoblastoma, Wilm's tumor, multiple myeloma; skin cancer(e.g., melanoma), lymphomas and blood cancer.

More preferably, an siRNA of the invention is used to inhibit choroidalneovascularization in age-related macular degeneration.

For treating angiogenic diseases, the siRNA of the invention canadministered to a subject in combination with a pharmaceutical agentwhich is different from the present siRNA. Alternatively, the siRNA ofthe invention can be administered to a subject in combination withanother therapeutic method designed to treat the angiogenic disease. Forexample, the siRNA of the invention can be administered in combinationwith therapeutic methods currently employed for treating cancer orpreventing tumor metastasis (e.g. radiation therapy, chemotherapy, andsurgery). For treating tumors, the siRNA of the invention is preferablyadministered to a subject in combination with radiation therapy, or incombination with chemotherapeutic agents such as cisplain, carboplatin,cyclophosphamide, 5-fluorouracil, adriamycin, daunorubicin or tamoxifen.

In the present methods, the present siRNA can be administered to thesubject either as naked siRNA, in conjunction with a delivery reagent,or as a recombinant plasmid or viral vector which expresses the siRNA.

Suitable delivery reagents for administration in conjunction with thepresent siRNA include the Mirus Transit TKO lipophilic reagent;lipofectin; lipofectamine; cellfectin; or polycations (e.g.,polylysine), or liposomes. A preferred delivery reagent is a liposome.

Liposomes can aid in the delivery of the siRNA to a particular tissue,such as retinal or tumor tissue, and can also increase the bloodhalf-life of the siRNA. Liposomes suitable for use in the invention areformed from standard vesicle-forming lipids, which generally includeneutral or negatively charged phospholipids and a sterol, such ascholesterol. The selection of lipids is generally guided byconsideration of factors such as the desired liposome size and half-lifeof the liposomes in the blood stream. A variety of methods are known forpreparing liposomes, for example as described in Szoka et al. (1980),Ann. Rev. Biophys. Bioeng. 9: 467; and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369, the entire disclosures of which areherein incorporated by reference.

Preferably, the liposomes encapsulating the present siRNA comprises aligand molecule that can target the liposome to a particular cell ortissue at or near the site of angiogenesis. Ligands which bind toreceptors prevalent in tumor or vascular endothelial cells, such asmonoclonal antibodies that bind to tumor antigens or endothelial cellsurface antigens, are preferred.

Particularly preferably, the liposomes encapsulating the present siRNAare modified so as to avoid clearance by the mononuclear macrophage andreticuloendothelial systems, for example by havingopsonization-inhibition moieties bound to the surface of the structure.In one embodiment a liposome of the invention can comprise bothopsonization-inhibition moieties and a ligand.

Opsonization-inhibiting moieties for use in preparing the liposomes ofthe invention are typically large hydrophilic polymers that are bound tothe liposome membrane. As used herein, an opsonization inhibiting moietyis “bound” to a liposome membrane when it is chemically or physicallyattached to the membrane, e.g., by the intercalation of a lipid-solubleanchor into the membrane itself, or by binding directly to active groupsof membrane lipids. These opsonization-inhibiting hydrophilic polymersform a protective surface layer which significantly decreases the uptakeof the liposomes by the macrophage-monocyte system (“MMS”) andreticuloendothelial system (“RES”); e.g., as described in U.S. Pat. No.4,920,016, the entire disclosure of which is herein incorporated byreference. Liposomes modifies with opsonization-inhibition moieties thusremain in the circulation much longer than unmodified liposomes. Forthis reason, such liposomes are sometimes called “stealth” liposomes.

Stealth liposomes are known to accumulate in tissues fed by porous or“leaky” microvasculature. Thus, target tissue characterized by suchmicrovasculature defects, for example solid tumors, will efficientlyaccumulate these liposomes; see Gabizon, et al. (1988), P.N.A.S., USA,18: 6949-53. In addition, the reduced uptake by the RES lowers thetoxicity of stealth liposomes by preventing significant accumulation inthe liver and spleen. Thus, liposomes of the invention that are modifiedwith opsonization-inhibition moieties can deliver the present siRNA totumor cells.

Opsonization inhibiting moieties suitable for modifying liposomes arepreferably water-soluble polymers with a number-average molecular weightfrom about 500 to about 40,000 daltons, and more preferably from about2,000 to about 20,000 daltons. Such polymers include polyethylene glycol(PEG) or polypropylene glycol (PPG) derivatives; e.g., methoxy PEG orPPG, and PEG or PPG stearate synthetic polymers such as polyacrylamideor poly N-vinyl pyrrolidone; linear, branched, or dendrimericpolyamidoamines; polyacrylic acids; polyalcohols, e.g.,polyvinylalchohol and polyxylitol to which carboxylic or amino groupsare chemically linked, as well as gangliosides, such as ganglioside GM₁.Copolymers of PEG, methoxy PEG, or methoxy PPG, or derivatives thereof,are also suitable. In addition, the opsonization inhibiting polymer canbe a block copolymer of PEG and either a polyamino acid, polysaccharide,polyamidoamine, polyethyleneamine, or polynucleotide. The opsonizationinhibiting polymers can also be natural polysaccharides containing aminoacids or carboxylic acids, e.g., galacturonic acid, glucuronic acid,mannuronic acid, hyaluronic acid, pectic acid, neuraminic acid, alginic,acid, carageenan; aminated polysaccharides or oligosaccharides (linearor branched); or carboxylated polysaccharides or oligosaccharides, e.g.,reacted with derivatives of carbonic acids with resultant linking ofcarboxylic groups.

Preferably, the opsonization-inhibiting moiety is a PEG, PPG, orderivatives thereof. Liposomes modified with PEG or PEG-derivatives aresometimes called “PEGylated liposomes.”

The opsonization inhibiting moiety can be bound to the liposome membraneby any one of numerous well-known techniques. For example anN-hydroxysuccinimide ester of PEG can be bound to aphosphatidyl-ethanolamine lipid-soluble anchor and then bound to amembrane. Similarily, a dextran polymer can be derivatized with astearylamine lipid-soluble anchor via reductive amination usingNa(CN)BH₃ and a solvent mixture such as tetrahydrofuran and water in a30:12 ratio at 60° C.

Recombinant plasmids which express siRNA of the invention are discussedabove. Such recombinant plasmids can also be administered directly or inconjunction with a suitable delivery reagent, including the MirusTransit LT1 lipophilic reagent; lipofetin; lipofectamine; cellfectin;polycations (e.g., polylysine) or liposomes. Recombinant viral vectorswhich express siRNA of the invention are also discussed above, andmethods for delivering such vectors to an area of neovascularization ina patient are within the skill in the art.

The siRNA of the invention can be administered to the subject by anymeans suitable for delivering the siRNA to the cells of the tissue at ornear the area of neovascularization. For example, the siRNA can beadministered by gene gun, eletroporation, or by other suitableparenteral or enteral administration routes.

Suitable enteral administration routes include oral, rectal orintranasal delivery.

Suitable parenteral administration routes include intravaseularadministration (e.g. intravenous bolus injection, intravenous infusion,intra-arterial bolus injection, intra-arterial infusion and catheterinstillation into the vasculature); peri- and intra-tissueadministration (e.g. peri-tumoral and intra-tumoral injection,intra-retinal injection or subretinal injection); subcutaneous injectionor deposition including subcutaneous infusion (such as by osmoticpumps); direct (e.g., topical) application to the area at or near thesite of neovascularization, for example by a catheter or other placementdevice (e.g., a corneal pellet or a suppository, eye-dropper, or animplant comprising a porous, non-porfous, or gelatinous material); andinhalation. Suitable placement devices include the ocular implantsdescribed in U.S. Pat. Nos. 5,902,598 and 6,375,972, and thebiodegradable ocular implants described in U.S. Pat. No. 6,331,313, theentire disclosures of which are herein incorporated by reference. Suchocular implants are available from (Control Delivery Systems, Inc.(Watertown, Mass.) and Oculex Pharmaceuticals, Inc. (Sunnyvale, Calif.).

In a preferred embodiment, injections or infusions of the siRNA aregiven at or near the site of neovascularization. More preferably, thesiRNA is administered topically to the eye, e.g. in liquid or gel formto the lower eye lid or conjunctival cul-de-sac, as is within the skillin the art (see, e.g., Acheampong AA et al., 2002, Drug Metabol. andDisposition 30: 421-429, the entire disclosure of which is hereinincorporated by reference).

Typically,, the siRNA of the invention is administered topically to theeye in amounts of from about 5 microliters to about 75 micro;iters, forexample from about 7 microliters to about 50 microliters, preferablyfrom about 1.0 microliters to about 30 microliters. It is understoodthat topical instillation in the eye of siRNA in volumes greater than 75microliters cane result in loss of siRNA from the eye through spillageand drainage. Thus, it is preferable to administer a high concentrationof siRNA (e.g., 100-1000 nM) in as small a volume as possible.

A particularly preferred parenteral administration route is intraocularadministration. It is understood that intraocular administration of thepresent siRNA can be accomplished by injection or direct (e.g., topical)administration to the eye, as long as the administration route allowsthe siRNA to enter the eye. In addition to the topical routes ofadministration to the eye described above, suitable intraocular routesof administration include intravitreal, intraretinal, subretinal,subtenon, peri- and retro-orbital, trans-corneal and trans-scleraladministration. Such intraocular administration routes are within theskill in the art; see, e.g., and Acheampong AA et al, 2002, supra; andBennett et al. (1996), Hum. Gene Ther: 7: 1763-1769 and Ambati J et at.al., 2002, Progress in Retinal and Eye Res. 21: 145-151, the entiredisclosures of which are herein incorporated by reference.

The siRNA of the invention can be administered in a single dose or inmultiple doses. Where the administration of the siRNA of the inventionis by infusion, the infusion can be a single sustained dose or can bedelivered by multiple infusions. Injection of the agent directly intothe tissue is at or near the site of neovascularization preferred.Multiple injections of the agent into the tissue at or near the site ofneovascularization are particularly preferred.

One skilled in the art can also readily determine an appropriate dosageregimen for administering the siRNA of the invention to a given subject.For example, the siRNA can be administered to the subject once, such asby a single injection or deposition at or near the neovascularizationsite. Alternatively, the siRNA can be administered to a subject multipletimes daily or weekly. For example, the siRNA can he administered to asubject once weekly for a period of from about three to abouttwenty-eight weeks, more preferably from about seven to about ten weeks.In a preferred dosage regimen, the siRNA is injected at or near the siteof neovascularization (e.g., intravirally) once a week for seven weeks.It is understood that periodic administrations of the siRNA of theinvention for an indefinite length of time may be necessary for subjectssuffering from a chronic neovascularization disease, such as wet ARMD ordiabetic retinopathy.

Where a dosage regimen comprises multiple administrations, it isunderstood that the effective amount of siRNA administered to thesubject can comprise the total amount of siRNA administered over theentire dosage regimen.

The siRNA of the invention are preferably formulated as pharmaceuticalcompositions prior to administering to a subject, according totechniques known in the art. Pharmaceutical compositions of the presentinvention are characterized as being at least sterile and pyrogen-free.As used herein, “pharmaceutical formulations” include formulations forhuman and veterinary use. Methods for preparing pharmaceuticalcompositions of the invention are within the skill in the art, forexample as described in Remington's Pharmaceutical Science, 17th ed.,Mack Publishing Company, Easton, Pa., (1985), the entire disclosure ofwhich is herein incorporated by reference.

The present pharmaceutical formulations comprise an siRNA of theinvention (e.g., 0.1 to 90% by weight), or a physiologically acceptablesalt thereof, mixed with a physiologically acceptable carrier medium.Preferred physiologically acceptable carrier media are water, bufferedwater, saline solutions (e.g., normal saline or balanced salinesolutions such as Hank's or Earle's balanced salt solutions), 0.4%saline, 0.3% glycine, hyaluronic acid and the like.

Pharmaceutical compositions of the invention can also compriseconventional pharmaceutical excipients and/or additives. Suitablepharmaceutical excipients include stabilizers, antioxicants, osmolalityadjucting agents, buffers, and pH adjusting agents. Suitable additivesinclude physiologically biocompatible buffers (e.g., tromethaminehydrochloride), additions of chelants (such as, for example, DTPA orDTPA-bisamide) or calcium chelate complexes (as for example calciumDTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodiumsalts (for example calcium chloride, calcium ascorbate, calciumgluconate or calcium lactate). Pharmaceutical compositions of theinvention can be packaged for use in liquid form, or can be lyophilized.

For topical administration to the eye, conventional intraocular deliveryreagents can be used. For example, pharmaceutical compositions of theinvention for topical intraocular delivery can comprise saline solutionsas described above, corneal penetration enhancers, insoluble particles,petrolatum or other gel-based ointments, polymers which undergo aviscosity increase upon instillation in the eye, or mucoadhesivepolymers. Preferably, the intraocular delivery reagent increases cornealpenetration, or prolongs preocular retention of the siRNA throughviscosity effects or by establishing physicochemical interactions withthe mucin layer covering the corneal epithelium.

Suitable insoluble particles for topical intraocular delivery includethe calcium phosphate particles described in U.S. Pat. No. 6,355,271 ofBell et al., the entire disclosure of which is herein incorporated byreference. Suitable polymers which undergo a viscosity increase uponinstillation in the eye include polyethlylenepolyoxypropylene blockcopolymers such as polyoxamer 407 (e.g., at a concentration of 25%),cellulose acetophthalate (e.g., at a concentation of 30%), or alow-acetyl gellan gum such as Gelrite® (available from CP Kelco,Wilmington, Del.). Suitable mucoadhesive polymers include hydroceolloidswith multiple hydrophilic functional groups such as carboxyl, hydroxyl,amide and/or sulfate groups; for example hydroxypropylcellulose,polyacrylic acid, high-molecular weight polyethylene glycols(e.g., >200,000 number average molecular weight), dextrans, hyaluronicacid, polygalacturonic acid, and xylocan. Suitable corneal penetrationenhancers include cyclodextrins, benzalkonium chloride, polyoxyethyleneglycol lauryl ether (e.g., Brij® 35), polyoxyethylene glycol stearylether (e.g., Brij® 78), polyoxyethylene glycol oleyl ether (e.g., Brij®98), ethylene diamine tetraacetic acid (EDTA), digitontin sodiumtaurocholate, saponins and polyoxyethylated castor oil such as CremaphorEL.

For solid compositions, conventional nontotxic solid carriers can beused; for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like.

For example, a solid pharmaceutical composition for oral administrationcan comprise any of the carriers and excipients listed above and 10-95%,preferably 25%-75%, of one or more siRNA of the invention. Apharmaceutical composition for aerosol (inhalational) administration cancomprise 0.01-20% by weight, preferably 1-10% by weight, of one or moresiRNA of the invention encapsulated in a liposome as described above,and propellant. A carrier can also be included as desired; e.g.,lecithin for intranasal delivery.

The invention will now be illustrated with the following non-limitingexamples. The animal experiments described in Examples 4-6 and 8-9 wereperformed using the University of Pennsylvania institutional guidelinesfor the care and use of animals in research. The animal experimentdescribed in Example 10 will be performed in accordance with theStandard Operating Procedures of Sierra Biomedical. 587 Dunn Circle,Sparks, Nev., 89431.

EXAMPLE 1 siRNA Transfection and Hypoxia Induction in Vitro

siRNA Design—A 19 nt sequence located 329 nt from the 5′ end of humanVEGF mRNA was chosen as a target sequence: AAACCTCACCAAGGCCAGCAC (SEQ IDNO: 51). To ensure that it was not contained in the mRNA from any othergenes, this target sequence was entered into the BLAST search engineprovided by NCBI. The use of the BLAST algorithm is described inAltschul et al. (1990), J. Mol. Biol. 215: 403-410 and Altschul et al.(1997), Nucleic Acids Res. 25: 3389-3402, the disclosures of which areherein incorporated by reference in their entirety. As no other mRNA wasfound which contained the target sequence, an siRNA duplex wassynthesized to target this sequence (Dharmacon Research, Inc., LafayetteColo.).

The siRNA duplex had the following sense and antisense strands. sense:5′-accucaccaaggccagcacTT-3′. (SEQ ID NO: 77) antisense:5′-gugcuggccuuggugagguTT-3′. (SEQ ID NO: 78)

Together, the siRNA sense and antisense strands formed a 19 ntdouble-stranded siRNA with TT3′ overhangs (shown in bold) on eachstrand. This siRNA was termed “Candidate 5” or “Cand5.” Other siRNAwhich target human VEGF mRNA were designed and tested as described forCand5.

An siRNA targeting the following sequence in green fluorescent protein(GFP) mRNA was used as a nonspecific control: GGCTACGTCCAGCGCACC (SEQ IDNO: 79). The siRNA was purchased from Dharmacon (Lafayette Colo.).

siRNA Transfection and Hypoxia Induction In Vitro—Human cell lines (293;Hela and ARPE19) were separately seeded into 24-well plates in 250microlitres of complete DMEM medium one day prior to transfection, sothat the cells were ˜50% confluent at the time of transfection. Cellswere transfected with 2.5 nM Cand5 siRNA, and with either no siRNA or2.5 nM non-specific siRNA (targeting GFP) as controls. Transfectionswere performed in all cell lines with the “Transit TKO Transfection”reagent, as recommended by the manufacturer (Mirus).

Twenty four hours after transfection, hypoxia was induced in the cellsby the addition of desferoxamide mesylate to a final concentration of130 micromolar in each well. Twenty four hours post-transfection, thecell culture medium was removed from all wells, and a human VEGF ELISA(R&D systems, Minneapolis Minn.) was performed on the culture medium asdescribed in the Quantikine human VEGF ELISA protocol available from themanufacturer, the entire disclosure of which is herein incorporated byreference.

As can be seen in FIG. 1, RNAi degradation induced by Cand5 siRNAsignificantly reduces the concentration of VEGF produced by the hypoxic293 and HeLa cells. There was essentially no difference in the amount ofVEGF produced by hypoxic cells treated with either no siRNA or thenon-specific siRNA control. Similar results were also seen with humanARPE19 cells treated under the same conditions. Thus, RNA interferencewith VEGF-targeted siRNA disrupts the pathogenic up-regulation of VEGFin human cultured cells in vitro.

The experiment outlined above was repeated on mouse NIH 3T3 cells usinga mouse-specific VEGF siRNA (see Example, 6 below), and VEGF productionwas quantified with a mouse VEGF ELISA (R&D systems, Minneapolis, Minn.)as described in the Quantikine mouse VEGF ELISA protocol available fromthe manufacturer, the entire disclosure of which is herein incorporatedby reference. Results similar to those reported in FIG. 1 for the humancell lines were obtained.

EXAMPLE 2 Effect of Increasing siRNA Concentration on VEGF Production inHuman Cultured Cells

The experiment outlined in Example 1 was repeated with human 293, HeLaand ARPE19 cells using a range of siRNA concentrations from 10 nM to 50nM. The ability of the Cand5 siRNA to down-regulate VEGF productionincreased moderately up to approximately 13 nM siRNA, but a plateaueffect was seen above this concentration. These results high light thecatalytic nature of siRNA-mediated RNAi degradation of mRNA, as theplateau effect appears to reflect VEGF production from the few cells nottransfected with the siRNA. For the majority of cells which had beentransfected with the siRNA, the increased VEGF mRNA production inducedby the hypoxia is outstripped by the siRNA-induced degradation of thetarget mRNA at siRNA concentrations greater than about 13 nM.

EXAMPLE 3 Specificity of siRNA Targeting

NIH 3T3 mouse fibroblasts were grown in 24-well plates under standardconditions, so that the cells were ˜50% confluent one day prior totransfection. The human VEGF siRNA Cand5 was transfected into a NIH 3T3mouse fibroblasts as in Example 1. Hypoxia was then induced in thetransfected cells, and murine VEGF concentrations were measured by ELISAas in Example 1.

The sequence targeted by the human VEGF siRNA Cand5 differs from themurine VEGF mRNA by one nucleotide. As can be seen in FIG. 2, the humanVEGF siRNA has no affect on the ability of the mouse cells toup-regulate mouse VEGF after hypoxia. These results show that siRNAinduced RNAi degradation is sequence-specific to within a one nucleotideresolution.

EXAMPLE 4 In Vivo Delivery of siRNA to Murine Retinal Pigment EpithelialCells

VEGF is upregulated in the retinal pigment epithelial (RPE) cells ofhuman patients with age-related macular degeneration (ARMD). To showthat functional siRNA can be delivered to RPE cells in vivo. GFP wasexpressed in mouse retinas with a recombinant adenovirus, and GSPexpression was silenced with siRNA. The experiment was conducted asfollows.

One eye from each of five adult C57/Black6 mice (Jackson Labs, BarHarbor, Me.) was injected subretinally as described in Bennett et al.(1996), supra with a mixture containing ˜1×10⁸ particles of adenoviruscontaining eGFP driven by the CMV promoter and 20 picomoles of siRNAtargeting eGFP conjugated with transit TKO reagent (Mirus).

As positive control, the the contralateral eyes were injected with amixture containing ˜1×10⁸ particles of adenovirus containing eGFP drivenby the CMV promoter and 20 picomoles of siRNA targeting human VEGFconjugated with transit TKO reagent (Mirus). Expression of GFP wasdetected by fundus ophthalmoscopy 48 hours and 60 hours after injection.Animals were sacrificed at either 48 hours or 60 hours post-injection.The eyes were enucleated and fixed in 4% parafomaldehyde, and wereprepared either as flat mounts or were processed into 10 microncryosections for fluorescent microscopy.

No GFP fluorescence was detectable by ophthalmoscopy in the eyes whichreceived the siRNA targeted to GFP mRNA in 4 out of 5 mice, whereas GFPfluorescence was detectable in the contralateral eye which received thenon-specific control siRNA. A representative flat mount analyzed byfluorescence microscopy showed a lack of GFP fluorescence in the eyewhich received GFP siRNA, as compared to an eye that received thenon-specific control siRNA. Cryosections of another retina showed thatthe recombinant adenovirus efficiently targets the RPE cells, and whenthe adenovirus is accompanied by siRNA targeted to GFP mRNA, expressionof the GFP transgene is halted.

While there is some GFP fluorescence detectable by fluorescencemicroscopy in eyes that received siRNA targeted to GFP mRNA, thefluorescence is greatly suppressed as compared to controls that receivednon-specific siRNA. These data demonstrate that functional siRNA can bedelivered in vivo to RPE cells.

EXAMPLE 5 In Vivo Expression and siRNA-Induced RNAi Degradation of HumanVEGF in Murine Retinas

In order to demonstrate that siRNA targeted to VEGF functioned in vivo,an exogenous human VEGF expression cassette was delivered to mouse RPEcells via an adenovirus by subretinal injection, as in Example 4. Oneeye received Cand5 siRNA, and the contralateral eye received siRNAtargeted to GFP mRNA. The animals were sacrificed 60 hourspost-injection, and the injected eyes were removed and snap frozen inliquid N₂ following enucleation. The eyes were then homogenized in lysisbuffer, and total protein was measured using a standard Bradford proteinassay (Roche, Germany). The samples were normalized for total proteinprior to assaying for human VEGF by ELISA as described in Example 1.

The expression of VEGF was somewhat variable from animal to animal. Thevariability of VEGF levels correlated well to those observed in the GFPexperiments of Example 4, and can be attributed to some error frominjection to injection, and the differential ability of adenovirus todelivery the target gene in each animal. However there was a significantattenuation of VEGF expression in each eye that received VEGF siRNA, ascompared to the eyes receiving the non-specific control siRNA (FIG. 4).These data indicate that the Cand5 siRNA was potent and effective insilencing human VEGF expression in murine RPE cells in vivo.

EXAMPLE 6 Inhibition of Choroidal Neovascularization in the Mouse CNVModel

There is evidence that choroidal neovascularization in ARMD is due tothe upregulation of VEGF in the RPE cells. This human pathologiccondition can be modeled in the mouse by using a laser to burn a spot onthe retina (“laser photo-coagulation” or “laser induction”). During thehealing process, VEGF is believed to be upregulated in the RPE cells ofthe burned region, leading to re-vascularization of the choroid. Thismodel is called the mouse choroidal neovascularization )“CNV”) model.

For rescue of the mouse CNV model, a mouse siRNA was designed thatincorporated a one nucleotide change from the human “Cand5” siRNA fromExample 1. The mouse siRNA specifically targeted mouse VEGF mRNA at thesequence AAACCUCACCAAAGCCAGCAC (SEQ ID NO: 80). Other siRNA that targetmouse VEGF were also designed and tested. The GFP siRNA used as anonspecific control in Example 1 was also used as a non-specific controlhere.

Twenty four hours after laser induction, one eye from each of elevenadult C57/Black6 mice (Jackson Labs, Bar Harbor, Me.) was injectedsubretinally with a mixture containing ˜1×10⁸ particles of adenoviruscontaining LacZ driven by the CMV promoter and 20 picomoles of siRNAtargeting mouse VEGF conjugated with transit TKO reagent (Mirus), as inExample 4. As a control, contralateral eyes received a mixturecontaining ˜1×10⁸ particles of adenovirus containing LacZ driven by theCMV promoter and 20 picomoles of siRNA targeting GFP conjugated withtransit TKO reagent (Mirus).

Fourteen days after the laser treatment, the mice were perfused withfluorescein and the area of neovascularization was measured around theburn spots. Areas of the burn spots in the contra-lateral eye were usedas a control. The site of neovascularization around the burn spots inanimals that received siRNA targeting mouse VEGF was, on average, ¼ thearea of the control areas. These data support the use of VEGF-directedsiRNA (also called “anti-VEGF siRNA”) for therapy of ARMD.

EXAMPLE 7 Generation of an Adeno-Associated Viral Vector for Expressionof siRNA

A “cis-acting” plasmid for generating a recombinant AAV vector fordelivering an siRNA of the invention was generated by PCR basedsubcloning, essentially as described in Samulski R et al. (1987) supra.The cis-acting plasmid was called “pAAVsiRNA.”

The rep and cap genes of psub201 were replaced with the followingsequences in this order: a 19 nt sense RNA strand coding sequence inoperable connection with a polyT termination sequence under the controlof a human U6 RNA promoter, and a 19 nt antisense RNA strand codingsequence in operable connection with a polyT termination sequence underthe control of a human U6 RNA promoter. A schematic representation ofpAAVsiRNA is given if FIG. 5.

A recombinant AAV siRNA vector was obtained by transfecting pAAVsiRNAinto human 293 cells previously infected with E1-deleted adenovirus, asdescribed in Fisher K J et al. (1996). supra. The AAV rep and capfunctions were provided by a trans-acting plasmid pAAV/Ad as describedin Samulski R et al. (1989), supra. Production lots of the recombinantAAV siRNA vector were titered according to the number of genomecopies/ml, as described in Fisher K J et al. (1996), supra.

EXAMPLE 8 VEGF-Directed siRNA Inhibits Experimental ChoroidalNeovascularization

The ability of murine VEGF-directed siRNA to inhibit experimentallaser-induced choroidal neovascularization (CNV) in mice was tested asfollows.

The retinas of adult female C57BL/6 mice were laser photocoagulatedusing an 810 nm diode laser (75 um, 140 mw, 0.10 seconds) (OcuLight Six;IRIS Medical, Mountain View, Calif.). Three laser spots were applied toboth eyes of each mouse. Thirty-six hours following laserphotocoagulation, an siRNA targeted to mouse VEGF (“mVEGF1.siRNA”) wasdelivered subretinally or intravitreally to one eye of each mouse. Forsubretinal injection, the siRNA was conjugated with Transit TKOtransfection reagent (Mirus) and mixed with recombinant adenovirus(rAdenovirus). For intravitreal injection, the siRNA was delivered inthe absence of transfection reagent and rAdenovirus. As a control, thecontralateral eyes of each mouse received subretinal or intravitrealinjections of identical formulations with an siRNA targeted to GFP(“GFP1.siRNA”), which has no homology to mouse VEGF.

Fourteen days following laser treatment, all animals were perfused withhigh molecular weight FITC-dextran, choroidal flat mounts were preparedas described above, and the flat mounts were photographed and analyzedmicroscopically in a masked fashion. The area of CNV in each flat mountwas measured with Openlab software (Improvision, Boston, Mass.). Themean areas of CNV in eyes treated with mVEGF1.siRNA were significantlysmaller than those areas from GFFP1.siRNA-treated eyes for bothsubretinal (FIG. 6A; P<0.003) and intravitreal (FIG. 6B; P<0.04)delivery.

In a second experiment, the retinas of adult female C57BL/6 mice werelaser photocoagulated as described above, and the animals were dividedinto control and test groups. One day following laser photocoagulation,phosphate buffered saline was delivered intravitreally to the animals ofthe control group, which were perfused with dextran-fluorescein 14 daysafter laser treatment. Choroidal flat mounts were then prepared and theareas of CNV in each flat mount were measured as above.

Fourteen days following laser photocoagulation, mVEGF1.siRNA wasdelivered by intravitreal injection into one eye of each mouse in thetest group. Contralateral eyes were injected with GFP1.siRNA as acontrol. The test group animals, were perfused with high molecularweight dextran-fluorescein 21 days after laser treatment. Choroidal flatmounts were then prepared and the areas of CNV in each flat mount weremeasured, as above.

In this latter experiment, the anti-VEGF siRNA was administered duringCNV growth, as opposed to before CNV growth, and thus is morerepresentative of the condition of human patients presenting with wetAMD. As can be seen from FIG. 6, the mean areas of CNV inmVEGF1.siRNA-treated eyes were significantly smaller than those areasmeasured in GFP1.siRNA-treated eyes (FIG. 6C; P<0.05). The mean areas ofCNV in mVEGF1.siRNA-treated eyes at day 21 and control (“PBS”) eyes atday 14 were not significantly different (FIG. 6C; P=0.469).

The results of these experiments indicate that age-related maculardegeneration can be treated with anti-VEGF siRNA.

EXAMPLE 9 In Vivo RNA Interference of Human VEGF Induced by anti-VEGFsiRNA in Murine RPE Cells

The ability of Cand5 siRNA to induce RNAi of VEGF in vivo over time wasevaluated as follows.

AAV.CMV.VEGF, which expresses human VEGF from an adeno-associated viralvector, was generously provided by Dr. A. Auricchio. AAV.CMV.VEGF wasinjected subretinally and bilaterally in eyes of five C57B1/6 mice.Twenty-eight days after injection of AAV.CMV.VEGF, Cand5 siRNA wasdelivered by intravitreal injection into one eye and control GFP1.siRNAwas delivered by intravitreal injection in the contralateral eye of eachanimal.

At day 0 (pre-siRNA injection) and at 6, 10 and 14 days after siRNAinjection, the mice, were sacrificed and the eyes were snap frozen inliquid nitrogen following enucleation. The eyes were then homogenized inlysis buffer (Roche, Basel, Switzerland), and total protein was measuredusing a Bradford assay, as in Example 5 above. Two mice were used forthe 0 day time point (n=2), and three mice each were used for the 6, 10and 14 day time points (n=3). The samples were normalized for totalprotein prior to assaying for human VEGF by ELISA, according themanufacturer's recommendations (R&D systems, Minneapolis, Minn.).Percent of VEGF (% VEGF) for each mouse was calculated as theconcentration of VEGF (“[VEGF]”) in the eye injected with Cand5 dividedby the [VEGF] in the eye injected with GFP1.siRNA, multiplied by 100.

As can be seen from FIG. 7, a single injection of Cand5 induced anRNAi-mediated decrease in VEGF levels approximately 70% by day 6post-siRNA injection, with a reduction in VEGF production ofapproximately 35% continuing through at least day 14 post-siRNAinjection. These results indicate that siRNA directed against human VEGFis capable of inducing RNAi of human VEGF in vivo for a sustained periodof time.

EXAMPLE 10 In Vivo RNA Interference of VEGF in Monkeys with Anti-VEGFsiRNA

It is expected that 10 naïve cynomolgus monkeys (Macaca fascicularis)will be subjected to laser photocoagulation of the retina in both eyesto induce choroidal neovascularization (“CNV”). The following representsthe intended experimental plan to evaluate the ability of Cand5 siRNA toreduce the area of CNV in the monkeys. As the VEGF RNA sequence targetedby Cand5 is identical in both Homo sapiens and M. fascicularis, Cand5 isexpected to induce RNAi of M. fascicularis VEGF RNA.

The experiment will be carried out by Sierra Biomedical (“SBi”), 587Dunn Circle, Sparks, Nev., 89431, in accordance with SBi's StandardOperating Procedures. All monkeys will undergo a full pre-study healthscreen consisting of a physical examination, hematologic andophthalmologic evaluations, serum chemistry, and electroretinography(“ERG”). A pre-study fluorescein angiography of the monkeys's eyes willalso be performed, and inraocular pressure will be measured pre-studyand at two additional time mounts during the study period.

On day zero, both eyes of all 10 monkeys will be laser photocoagulatedto induce choroidal neovascularization. The animals will receivetwice-daily cageside observations and a once daily qualitativeassessment of food consumption throughout the experiment. At 14 dayspost laser induction, the monkeys will receive intravitreally one 50microliter dose of a Treatment or Control formulation (see below) ineach eye, in a randomized, double-blind fashion. For example, a givenanimal could receive a Treatment 1 dose in the right eye, and a Controldose in the left eye. Another animal could receive a Treatment 1 dose inthe right eye, and a Treatment 3 dose in the left eye. It is expectedthat four eyes will receive each formulation, for a total of 1 controland four treatment groups of four eyes each.

The formulations will be: Control—balanced saline solution (“BSS”)alone; Treatment 1—1 mg/ml Cand5 in BSS; Treatment 2—2.5 mg/ml Cand5 inBSS; Treatment 3—5 mg/ml Cand5 in BSS; and Treatment 4—10 mg/ml Cand5 inBSS. Prior to injection, it is expected that the pH, osmolarity, and theabsorbance at 260 nm of each formulation will be measured.

After laser induction, a fluorescein angiography will be performed onboth eyes of each monkey weekly up through the seventh week after laserinduction. The monkeys will then be sacrificed, and a complete necropsywill be performed with a full tissue collection (˜45 tissues including avitreous sample) and histopathologic evaluation of the collectedtissues. An ERG will be taken pre-necropsy.

It is expected that the laser-induced areas of CNV will be reduced uponadministration of the Cand5 siRNA, with the CNV area decreasing withincreasing dose of Cand5. However, a plateau effect such as is describedabove in Example 2 may be observed. The Cand5 siRNA is not expected toeffect any other organ or tissue except the choroid in the eye.

1. An isolated siRNA comprising a sense RNA strand and an antisense RNAstrand, wherein the sense and an antisense RNA strands form an RNAduplex, and wherein the sense RNA strand comprises a nucleotide sequenceidentical to a target sequence of about 19 to about 25 contiguousnucleotides in human VEGF mRNA, human Flt-1 mRNA, or human Flk-1/KDRmRNA, or an alternative splice form, mutant or cognate thereof.
 2. ThesiRNA of claim 1, wherein the human VEGF mRNA is selected from the groupconsisting of VEGF₁₂₁mRNA (SEQ ID NO: 2) VEGF₁₆₅mRNA (SEQ ID NO: 3);VEGF₁₈₉mRNA (SEQ ID NO: 4) and VEGF₂₀₆mRNA (SEQ ID NO: 5).
 3. The siRNAof claim 1 wherein the cognate of the human VEGF mRNA sequence is mouseVEGF mRNA (SEQ ID NO: 1).
 4. The siRNA of claim 1, wherein the sense RNAstrand comprises SEQ ID NO: 77, and the antisense strand comprises SEQID NO:
 78. 5. The siRNA of claim 1, wherein the sense RNA strandcomprises one RNA molecule and the antisense RNA strand comprises oneRNA molecule.
 6. The siRNA of claim 1, wherein the sense and antisenseRNA strands forming the RNA duplex are covalently linked by asingle-stranded hairpin.
 7. The siRNA of claim 1, wherein the siRNAfurther comprises non-nucleotide material.
 8. The siRNA of claim 1,wherein the sense and antisense RNA strands are stabilized againstnuclease degradation.
 9. The siRNA of claim 1, further comprising a 3′overhangs.
 10. The siRNA of claim 9, wherein the 3′ overhang comprisesfrom 1 to about 6 nucleotides.
 11. The siRNA of claim 9, wherein the 3′overhang comprises about 2 nucleotides.
 12. The siRNA of claim 5,wherein the sense RNA strand comprises a first 3′ overhang, and theantisense RNA strand comprises a second 3′ overhang.
 13. The siRNA ofclaim 12, wherein the first and second 3′ overhangs separately
 14. ThesiRNA of claim 13, wherein the first 3′ overhang comprises adinucleotide and the second 3′ overhang comprises a dinucleotide. 15.The siRNA of claim 14, wherein the dinucleotide comprising the first andsecond 3′ overhangs is dithymidylic acid (TT) or diuridylic acid (uu).16. The siRNA of claim 9, wherein the 3′ overhang is stabilized againstnuclease degradation.
 17. A retinal pigment epithelial cell comprisingthe siRNA of claim
 1. 18. A recombinant plasmid comprising nucleic acidsequences for expressing an siRNA comprising a sense RNA strand and anantisense RNA strand, wherein the sense and an antisense RNA strandsform an RNA duplex, and wherein the sense RNA strand comprises anucleotide sequence identical to a target sequence of about 19 to about25 contiguous nucleotides in human VEGF mRNA, human Flt-1 mRNA, or humanFlk-1/KDR mRNA, or an alternative splice form, mutant or cognatethereof.
 19. The recombinant plasmid of claim 18, wherein the nucleicacid sequences for expressing the siRNA comprise an inducible orregulatable promoter.
 20. The recombinant plasmid of claim 18, whereinthe nucleic acid sequences for expressing the siRNA comprise a sense RNAstrand coding sequence in operable connection with a polyT terminationsequence under the control of a human U6 RNA promoter, and an antisenseRNA strand coding sequence in operable connection with a polyTtermination sequence under the control of a human U6 RNA promoter. 21.The recombinant plasmid of claim 20, wherein the plasmid is pAAV siRNA.22. A recombinant viral vector comprising nucleic acid sequences forexpressing an siRNA comprising a sense RNA strand and an antisense RNAstrand, wherein the sense and an anti sense RNA strands from an RNAduplex, and wherein the sense RNA strand comprises a nucleotide sequenceidentical to a target sequence of about 19 to about 25 contiguousnucleotides in human VEGF mRNA, human Flt-1 mRNA, or human Flk-1/KDRmRNA, or an alternative splice form, mutant or cognate thereof.
 23. Therecombinant viral vector of claim 22, wherein the nucleic acid sequencesfor expressing the siRNA comprise an inducible or regulatable promoter.24. The recombinant viral vector of claim 22, wherein the nucleic acidsequences for expressing the siRNA comprise a sense RNA strand codingsequence in operable connection with a polyT termination sequence underthe control of a human U6 RNA promoter, and an antisense RNA strandcoding sequence in operable connection with a polyT termination sequenceunder the control of a human U6 RNA promoter.
 25. The recombinant viralvector of claim 22, wherein the recombinant viral vector is selectedfrom the group consisting of an adenoviral vector, an adeno-associatedviral vector, a lentiviral vector, a retroviral vector and a herpesvirus vector.
 26. The recombinant viral vector of claim 22, wherein therecombinant viral vector is pseudotyped with surface proteins fromvesicular stomatitis virus, rabies virus, Ebola virus, or Mokola virus.27. The recombinant viral vector of claim 25, wherein the recombinantviral vector comprises an adeno-associated viral vector.
 28. Apharmaceutical composition comprising an siRNA and a pharmaceuticallyacceptable carrier, wherein the siRNA comprises a sense RNA strand andan antisense RNA strand, wherein the sense and an antisense RNA strandsform an RNA duplex, and wherein the sense RNA strand comprises anucleotide sequence identical to a target sequence of about 19 to about25 contiguous nucleotides in human VEGF mRNA, human Flt-1 mRNA, or humanFlk-1/KDR mRNA, or an alternative splice form, mutant or cognatethereof.
 29. The pharmaceutical composition of claim 8, furthercomprising lipofectin, lipofectamine, cellfectin, polycations, orliposomes.
 30. A pharmaceutical composition comprising the plasmid ofclaim 18, or a physiologically acceptable salt thereof, and apharmaceutically acceptable carrier.
 31. The pharmaceutical compositionof claim 30, further comprising lipofectin, lipofectamine, cellfectin,polycations, or liposomes.
 32. A pharmaceutical composition comprisingthe viral vector of claim 22 and a pharmaceutically acceptable carrier.33. A method of inhibiting expression of human VEGF mRNA, human Flt-1mRNA, or human Flk-1/KDR mRNA, or an alternative splice form, mutant orcognate thereof, comprising administering to a subject an effectiveamount of an siRNA comprising a sense RNA strand and a antisense RNAstrand, wherein the sense and an antisense RNA strands form an RNAduplex, and wherein the sense RNA strand comprises a nucleotide sequenceidentical to a target sequence of about 19 to about 25 contiguousnucleotides in human VEGF mRNA, human Flt-1 mRNA, or human Flk-1/KDRmRNA, or an alternative splice form, mutant or cognate thereof, suchthat the human VEGF mRNA, human Flt-1 mRNA, or human Flk-1/KDR mRNA, oran alternative splice form, mutant or cognate thereof, is degraded. 34.The method of claim 33, wherein the subject is a human being.
 35. Themethod of claim 33, wherein expression of human VEGF mRNA, human Flt-1mRNA, or human Flk-1/KDR mRNA, or an alternative splice form, mutant orcognate thereof is inhibited in one or both eyes of the subject.
 36. Themethod of claim 33 wherein expression of human VEGF mRNA, human Flt-1mRNA, or human Flk-1/KDR mRNA or an alternative splice form, mutant orcognate thereof is inhibited in retinal pigment epithelial cells of thesubject.
 37. The method of claim 33, wherein the effective amount of thesiRNA is from about 1 nM to about 100 nM.
 38. The method of claim 33,wherein the siRNA is administered in conjunction with a deliveryreagent.
 39. The method of claim 38, wherein the delivery agent isselected from the group consisting of lipofectin, lipofectamine,cellfectin, polycations, and liposomes.
 40. The method of claim 39,wherein the delivery agent is a liposome.
 41. The method claim 40,wherein the liposome comprises a ligand which targets the liposome tocells at or near the site of angiogenesis.
 42. The method of claim 41,wherein the ligand binds to receptors on tumor cells or vascularendothelial cells.
 43. The method of claim 42, wherein the ligandcomprises a monoclonal antibody.
 44. The method of claim 40, wherein theliposome is modified with an opsonization-inhibition moiety.
 45. Themethod of claim 44, wherein the opsonization-inhibiting moiety comprisesa PEG, PPG, or derivatives thereof.
 46. The method of claim 33, whereinthe siRNA is expressed from a recombinant plasmid
 47. The method ofclaim 33, wherein the siRNA is expressed from a recombinant viralvector.
 48. The method of claim 47, wherein the recombinant viral vectorcomprises an adenoviral vector, an adeno-associated viral vector, alentiviral vector, a retroviral vector, or a herpes virus vector. 49.The method of claim 48, wherein the recombinant viral vector ispseudotyped with surface proteins from vesicular stomatitis virus,rabies virus, Ebola virus, or Mokola virus.
 50. The method of claim 47,wherein the recombinant viral vector comprises an adeno-associated viralvector.
 51. The method of claim 33, wherein the siRNA is administered byan enteral administration route.
 52. The method of claim 51, wherein theenteral administration route is selected from the group consisting oforal, rectal, and intranasal.
 53. The method of claim 33, wherein thesiRNA is administered by a parenteral administration route.
 54. Themethod of claim 53, wherein the parenteral administration route isselected from the group consisting of intravascular administration,peri- and intra-tissue administration, subcutaneous injection ordeposition, subcutaneous infusion, intraocular administration, anddirect application at or near the site of neovascularization.
 55. Themethod of claim 54, wherein the intravascular administration is selectedfrom the group consisting of intravenous bolus injection, intravenousinfusion, intra-arterial bolus injection, intra-arterial infusion andcatheter instillation into the vasculature.
 56. The method of claim 54,wherein the peri- and intra-tissue injection comprises peri-tumoralinjection or intra-tumoral injection.
 57. The method of claim 54,wherein the intraocular administration comprises intravitreal,intraretinal, subretinal, subtenon, peri- and retro-orbitaltrans-corneal or trans-scleral administration.
 58. The method of claim54, wherein the direct application at or near the site ofneovascularization comprises application by catheter, corneal pellet,eye dropper, suppository, an implant comprising a porous material, animplant comprising a non-porous material, or an implant comprising agelatinous material.
 59. The method of claim 54, wherein the site ofneovascularization is in the eye, and the direct application at or nearthe site of neovascularization comprises application by an ocularimplant.
 60. The method of claim 59 wherein the ocular implant isbiodegradable.
 61. A method of inhibiting angiogenesis in a subject,comprising administering to a subject an effective amount of an siRNAcomprising a sense RNA strand and an antisense RNA strand, wherein thesense and an antisense RNA strands form an RNA duplex, and wherein thesense RNA, strand comprises a nucleotide sequence identical to a targetsequence of about 19 to about 25 contiguous nucleotides in human VEGFmRNA, human Flt-1 mRNA, or human Flk-1/KDR mRNA, or an alternativesplice form, mutant or cognate thereof.
 62. The method of claim 61,wherein the angiogenesis is pathogenic.
 63. The method of claim 61,wherein the angiogenesis is non-pathogenic.
 64. The method of claim 63,wherein the non-pathogenic angiogenesis is associated with production offatty tissues or cholesterol production.
 65. The method of claim 63,wherein the non-pathogenic angiogenesis comprises endometrialneovascularization.
 66. The method of claim 61, wherein the angiogenesisis inhibited in one or both eyes of the subject.
 67. The method of claim61, wherein the angiogenesis is inhibited in retinal pigment epithelialcells of the subject.
 68. A method of treating an angiogenic disease ina subject, comprising administering to a subject in need of suchtreatment an effective amount of an siRNA comprising a sense RNA strandand an antisense RNA strand, wherein the sense and an antisense RNAstrands form an RNA duplex, and wherein the sense RNA strand comprises anucleotide sequence identical to a target sequence of about 19 to about25 contiguous nucleotides in human VEGF mRNA, human Flt-1 mRNA, or humanFlk-1/KDR mRNA, or an alternative splice form, mutant or cognatethereof, such that angiogenesis associated with the angiogenic diseaseis inhibited.
 69. The method of claim 68, wherein the angiogenic diseasecomprises a tumor associated with a cancer.
 70. The method of claim 69,wherein the cancer is selected from the group consisting of breastcancer, lung cancer, head and neck cancer, brain cancer, abdominalcancer, colon cancer, colorectal cancer, esophagus cancer,gastrointestinal cancer, glioma, liver cancer, tongue cancerneuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostatecancer, retinoblastoma, Wilm's tumor, multiple mycloma, skin cancer,lymphoma, and blood cancer.
 71. The method of claim 68, wherein theangiogenic disease is selected from the group consisting of diabeticretinopathy, age-related macular degeneration, and inflammatorydiseases.
 72. The method of claim 71, wherein the inflammatory diseaseis psoriasis or rheumatoid arthritis.
 73. The method of claim 71,wherein the angiogenic disease is age-related macular degeneration. 74.The method of claim 68, wherein the siRNA is administered in combinationwith a pharmaceutical agent for treating the angiogenic disease, whichpharmaceutical agent is different from the siRNA.
 75. The method ofclaim 74, wherein the angiogenic disease is cancer, and thepharmaceutical agent comprises a chemotherapeutic agent.
 76. The methodof claim 74, wherein the chemotherapeutic agent is selected from thegroup consisting of cisplatin, carboplatin, cyclophosphamide,5-fluorouracil, adriamycin, daunorubicin, and tamoxifen.
 77. The methodof claim 68, wherein the siRNA is administered to a subject incombination with another therapeutic method designed to treat theangiogenic disease.
 78. The method of claim 77, wherein the angiogenicdisease is cancer, and the siRNA is administered in combination withradiation therapy, chemotherapy or surgery.